Biochemical analysis of siRNA/miRNA function in human

项目描述（由申请人提供）：本项目的主要目标是在分子水平上分析人类细胞中双链RNA （dsRNA）控制的基因表达调控机制。dsRNA分子可以由病毒感染或转座子活性产生，也可以通过编码短反向重复序列（microrna）的细胞基因以受调控的方式表达。将dsRNA引入与细胞基因同源的细胞已经彻底改变了细胞生物学研究，使研究人员能够抑制他们喜欢的基因进行功能分析，并为基因特异性治疗的新方法打开了大门。本项目的目的是鉴定和表征参与RNA沉默的蛋白质成分，并在分子水平上了解沉默活性核糖核蛋白复合物的形成和发挥其功能。1. 人siRNA/ mirna -蛋白复合物的表征。

DESCRIPTION (provided by applicant): The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of gene expression controlled by double-stranded RNA (dsRNA) in human cells. Molecules of dsRNA are either produced by viral infection or transposon activity, or they are expressed in a regulated manner from cellular genes that encode short inverted repeat sequences (microRNAs). Introduction of dsRNA into cells cognate to cellular genes has revolutionized cell biological studies enabling investigators to suppress their favorite gene for functional analysis and has opened the doors to new ways of gene-specific therapy. The aim of this project is to identify and characterize the protein components involved in RNA silencing and to understand at molecular level how silencing-active ribonucleoprotein complexes are formed and exert their function. 1. Characterization of the human siRNA/miRNA-protein complexes. 1.1 Investigate the role of siRNA/miRNA-associated Argonaute protein members (elF2C family, HIWI, HILl) and define their interaction network with cellular components required for RNAi and miRNA-mediated gene regulation. 1.2 Reconstitute the sequence-specific endonuclease complex (RISC) from recombinant proteins and short duplex or single-stranded siRNA molecules. 2. Isolation of miRNP/target mRNA complexes and identification of the cellular mRNAs subjected to microRNA-mediated control.